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The invention relates to methods and compositions for generating, modifying, adapting, and optimizing polynucleotide sequences that encode proteins having ADPGPP enzyme activities which are useful for introduction into plant species, and other hosts, and related aspects.
Genetic engineering of agricultural organisms dates back thousands of years to the dawn of agriculture. The hand of man has selected the agricultural organisms having the phenotypic traits that were deemed desirable, which desired phenotypic traits have often been taste, high yield, caloric value, ease of propagation, resistance to pests and disease, and appearance. Classical breeding methods to select for germplasm encoding desirable agricultural traits had been a standard practice of the world""s farmers long before Gregor Mendel and others identified the basic rules of segregation and selection. For the most part, the fundamental process underlying the generation and selection of desired traits was the natural mutation frequency and recombination rates of the organisms, which are quite slow compared to the human lifespan and make it difficult to use conventional methods of breeding to rapidly obtain or optimize desired traits in an organism.
The very recent advent of non-classical, or xe2x80x9crecombinantxe2x80x9d genetic engineering techniques has provided a new means to expedite the generation of agricultural organisms having desired traits that provide an economic, ecological, nutritional, or aesthetic benefit. To date most recombinant approaches have involved transferring a novel or modified gene into the germline of an organism to effect its expression or to inhibit the expression of the endogenous homologue gene in the organism""s native genome. However, the currently used recombinant techniques are generally unsuited for substantially increasing the rate at which a novel or improved phenotypic trait can be evolved. Essentially all recombinant genes in use today for agriculture are obtained from the germplasm of existing plant and microbial specimens, which have naturally evolved coordinately with constraints related to other aspects of the organism""s evolution and typically are not optimized for the desired phenotype(s). The sequence diversity available is limited by the natural genetic variability within the existing specimen gene pool, although crude mutagenic approaches have been used to add to the natural variability in the gene pool.
Unfortunately, the induction of mutations to generate diversity often requires chemical mutagenesis, radiation mutagenesis, tissue culture techniques, or mutagenic genetic stocks. These methods provide means for increasing genetic variability in the desired genes, but frequently produce deleterious mutations in many other genes. These other traits may be removed, in some instances, by further genetic manipulation (e.g., backcrossing), but such work is generally both expensive and time consuming. For example, in the flower business, the properties of stem strength and length, disease resistance and maintaining quality are important, but often initially compromised in the mutagenesis process.
The biosynthesis of starches in higher plants occurs in three steps, the first of which involves synthesis of ADP glucose from ATP and xcex1-glucose-1-phosphate, and which is catalyzed by ADP-glucose pyrophosphorylase (xe2x80x9cADPGPPxe2x80x9d; EC 2.7.7.27). The second step of starch biosynthesis is transfer of a glucosyl moiety of ADP-glucose to a maltodextrin or starch to give rise to a new xcex1-1,4-glucosyl linkage; the reaction is catalyzed by a starch synthase, of which there are several forms present either as soluble enzymes or bound to starch particles as particulate enzymes. The third reaction is catalyzed by branching enzyme and is responsible for synthesis of xcex1-1,6-glucosyl linkages.
Starch synthesis in plants is tightly regulated and is tied to photosynthetic carbon fixation. Principal control of starch synthesis in plants, algae, and bacteria is at the level of ADPGPP. It has been shown that reduced ADPGPP activity in Arabidopsis leaves and potato tubers results in a reduced rate of starch synthesis. The ADPGPP enzyme in plants exists primarily as a tetramer, S2L2, composed of two different subunits of approximately 50-60 kDa each. The molecular weight of the small (S) subunit is approximately 50-55 kDa, and the S subunit is the catalytic protein having the enzymatic active site (e.g., reaction center). The molecular weight of the large (L) subunit is approximately 55-60 kDa, and the L subunit is the regulatory subunit protein. The plant enzyme is strongly activated by 3-phosphoglycerate (PGA), a product of carbon dioxide fixation; in the absence of PGA, the enzyme exhibits only about 3% of its activity. Plant ADPGPP is also strongly inhibited by inorganic phosphate (Pi). In contrast, bacterial and algal ADPGPP exist as homotetramers of 50kDa. The Algal enzyme, like its plant counterpart, is activated by PGA and inhibited by Pi, whereas the bacterial enzyme is activated by fructose-1, 6-bisphosphate (FBP) and inhibited by AMP and Pi.
In the last 10 years, the demand for starch has dramatically increased both for food and industrial uses, primarily as a result of increased demand for high fructose corn syrups and biofuel. Hence, mobilizing a greater proportion of the photosynthetic assimilates of major crops into the seeds and other sinks in the form of starch can be expected to have a major impact on agriculture in the form of increased yield of harvestable parts. Deregulating starch biosynthesis by deregulating ADPGPP (e.g., decoupling from the need for positive activation and/or negative inhibition of catalytic activity) in order to increase both the rate of accumulation and the amount of starch in sinks such as tubers (e.g., potato) and seeds (e.g., maize, wheat, rice). A mutant form of E. coli ADPGPP gene (Gig C16) has been introduced into potato and exhibits a significant activity in the absence of its normal activator, FGP, and is much less sensitive to feedback inhibition by AMP and Pi. Transgenic potato plants expressing this gene under the control of a tuber-specific promoter showed 25-60% more starch in tubers as compared to control non-transgenic plants.
As noted, the advent of recombinant DNA technology has provided agriculturists with additional means of modifying plant genomes. While certainly practical in some areas to date genetic engineering methods have had limited success in transferring or modifying important biosynthetic or other pathways including the ADPGPP enzyme in photosynthetic organisms and bacteria. The creation of plants and other photosynthetic organisms having improved ADPGPP biosynthetic pathways can provide increased yields of certain types of starchy foodstuffs, enhanced biomass energy sources, and may alter the types and amounts of nutrients present in certain foodstuffs, among other desirable phenotypes.
Thus, there exists a need for improved methods for producing plants and agricultural photosynthetic microbes with an improved ADPGPP enzyme. In particular, these methods should provide general means for producing novel ADPGPP enzymes, including increasing the diversity of the ADPGPP gene pool and the rate at which genetic sequences encoding one or more ADPGPP subunits having desired properties are evolved. It is particularly desirable to have methods which are suitable for rapid evolution of genetic sequences to function in one or more plant species and confer an improved ADPGPP phenotype (e.g., reduced sensitivity to inhibitors (e.g., Pi, AMP), reduced dependence on activators (e.g., PGA, FBP), improved catalytic efficiency via increasing Vmax and/or increasing the apparent affinity of substrates for the enzyme, and/or relieving a requirement for allosteric activation or inhibition by allosteric repression, as well as plants which express the novel ADPGPP genetic sequence(s).
The present invention meets these and other needs and provides such improvements and opportunities.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention. All publications cited are incorporated herein by reference, whether specifically noted as such or not.
In a broad general aspect, the present invention provides a method for the rapid evolution of one or more polynucleotide sequences encoding a ADPGPP enzyme, or one or more subunits thereof; that, when transferred into an appropriate plant cell, or photosynthetic microbial host and expressed therein, confers an enhanced metabolic phenotype to the host to increase starch formation ratio and/or rate, or to increase the accumulation or depletion of certain starches. In general, polynucleotide sequence shuffling and phenotype selection, such as detection of a parameter of ADPGPP enzyme activity, is employed recursively to generate polynucleotide sequences which encode novel proteins having desirable ADPGPP enzymatic catalytic function(s), regulatory function(s), and related enzymatic and physicochemical properties. Although the method is believed broadly applicable to evolving biosynthetic enzymes having desired properties, the invention is described principally with reference to the metabolic enzyme activities of plants and/or photosynthetic microbes and/or bacteria, defined as ADPGPP, or an isozyme thereof, including both catalytic subunit (small subunit, S; gene designation, S) and allosteric regulatory subunit (large subunit, L; gene designation, L), respectively, as appropriate for plant and algal (S2L2), as well as bacterial (S4).
In one aspect, the invention provides methods of producing a recombinant cell having an elevated starch production activity. In the methods, one or more first ADGPP enzyme coding nucleic acid, or a homologue thereof, is recombined with one or more homologous first nucleic acid to produce a library of recombinant first enzyme nucleic acid homologues. This step can be repeated as desired to produce a more diverse library of recombinant first enzyme nucleic acid homologues. The libraries are selected for an activity which aids in Starch production, such as an increased or decreased catalytic rate, an altered substrate specificity, an increased ability of a cell expressing one or more members of the library to produce starch when the one or more library members is expressed in the cell, etc., thereby producing a selected library of recombinant first enzyme nucleic acid homologues. These steps are recursively repeated until one or more members of the selected library produces an elevated starch production level in a target recombinant cell when the one or more selected library member is expressed in the target cell, as compared to a starch fixation activity of the target cell when the one or more selected library member is not expressed in the target cell.